Summary Zika virus is a flavivirus that was first isolated in Uganda in 1947, and gained international notoriety during the 2015-2016 epidemic that spread to South America, North America, Pacific islands, and beyond (1-3). Previously associated with a mild or asymptomatic infection transmitted by Aedes mosquitos, the American Zika virus outbreak was associated with a greater incidence of microcephaly and Guillain Barr syndrome in the children of women infected during pregnancy. Evidence for both sexual and asexual transmission of Zika virus coupled with long latency periods continue to fuel public safety concerns. While experimental vaccination strategies are being pursued, there are no known cures for Zika virus infection or specific prophylactic treatments available for high risk professionals or individuals traveling to afflicted regions. Recent studies of molecular pathogenesis and virulence factors used by Zika virus and other flaviviruses have uncovered a general ability to disrupt or evade innate antiviral immune responses, primarily those mediated by type I and type III interferon (IFN), making the viruses more resistant to exogenous IFN therapy and contributing to virulence, tissue penetration, and host tropism. The flavivirus NS5 protein has been recognized as a primary antagonist of IFN-JAK-STAT signaling, and both Dengue virus and Zika virus have been shown to use NS5 to engage and destroy STAT2, an essential transcription regulator in the IFN response. While Dengue virus uses a specific cellular ubiquitin ligase enzyme to target STAT2, Zika virus uses a distinct mechanism mediated by unknown cellular machinery (13). Preliminary studies of Zika-mediated STAT2 degradation and IFN evasion in human cells provided a foundation for the design of sensitive living cell-based assay systems to identify cellular components used for STAT2 degradation and Zika virus replication. As mechanisms of virus host evasion are hypothesized to be high potential targets for therapeutic intervention, two complementary aims are proposed to (Aim 1) identify the cellular components required Zika NS5-mediated STAT2 degradation and (Aim 2) exploit this pathway as a target for small molecule inhibition of Zika replication. Focusing initial hits from these innovative experiments with general and mechanistic counter-screening and low-throughput follow-up analysis will reveal missing mechanistic machinery needed for Zika virus-mediated IFN evasion, provide cellular targets for investigations of virulence and pathogenesis to inform therapeutic interventions, and identify new leads for Zika virus antiviral compounds.